A High-Throughput Screening Approach To Repurpose FDA-Approved Drugs for Bactericidal Applications against Staphylococcus aureus Small-Colony Variants

FIG 1. Validation of the 22 screening hits against S. aureus small-colony variants (SCV). The 22 identified potential drug candidates that exhibited an adenylate kinase (AK) signal that surpassed hit selection criteria were assessed for antimicrobial efficacy against SCV at 100 μM. Of the 22 hits, 4 drugs, daunorubicin, ketoconazole, rifapentine, and sitafloxacin, demonstrated a ≥5-log reduction of viable bacteria in comparison to a mock-treated control, such that daunorubicin, rifapentine, and ketoconazole came back culture negative. Assay was performed in duplicate. * indicates culture-negative. Data presented as mean ± standard deviation.

FIG 2. Dose response of gentamicin, daunorubicin, ketoconazole, rifapentine, and sitafloxacin against established S. aureus biofilm using the MBEC assay. UAMS-1 biofilm was established on polystyrene pegs after 24 h of incubation with media supplemented with 10% (vol/vol) human plasma, and representative SEM is shown with magnifications of ×100 (A), ×1,000 (B), and ×5,000 (C). Biofilm was challenged by 1 to 128 µg/ml of each of the 4 validated HTS drugs and gentamicin (D). Sitafloxacin significantly killed biofilm at concentrations 4 to 128 µg/ml in comparison to gentamicin at each concentration. * indicates P < 0.005; # indicates P < 0.001 for the effect of gentamicin versus daunorubicin, ketoconazole, rifapentine, or sitafloxacin at each of the respective concentrations by 2-way ANOVA with Sidak’s test for post hoc comparisons. n = 3/group. Data presented as mean ± standard deviation.

FIG 3. Cytotoxicity of gentamicin, daunorubicin, ketoconazole, rifapentine, and sitafloxacin against human embryonic kidney (HEK) 293T cells. Gentamicin and each of the 4 validated screening hits were screened for cytotoxic effects at concentrations 1 to 128 mg/ml against HEK 293T cells using an XTT proliferation assay. Significance was judged for the effect of each drug concentration versus the mock-treated control by 2-way ANOVA with Sidak’s test for post hoc comparisons. * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.005, and **** indicates P < 0.001. n = 3/group. Data presented as mean ± standard deviation. Negative control was 64 mg/ml of zinc pyrithione.